Method of producing recombinant antibodies

ABSTRACT

The invention relates to novel nucleic acid sequences which encode an antibody suitable in the field of tumor diagnostics and therapeutics. Further, a method of producing recombinant antibodies is provided, wherein the novel nucleic acid sequencs are employed.

[0001] The invention relates to novel nucleic acid sequences whichencode an antibody suitable in the field of tumor diagnostics andtherapeutics. Further, a method of producing recombinant antibodies isprovided, wherein the novel nucleic acid sequencs are employed.

[0002] The monoclonal antibody G250, subclass IgG1, recognizes anantigen preferentially expressed on membranes of renal cell carcinomacells (RCC) and not expressed in normal proximal tubular epithelium. Theantibody G250 was obtained by immunizing a mouse with cell homogenatesfrom primary RCC lesions obtained from different patients (Oosterwijk etal., Int. J. Cancer 38 (1986), 489-494).

[0003] The antibody G250 as well as chimeric derivatives has been usedin clinical studies (Steffens et al., J. Clin. Oncol. 15 (1997),1529-1537). The nucleic acid sequence coding for the antigen-bindingsite of G250, however, has not been published yet.

[0004] Thus, a subject matter of the present invention is a nucleic acidencoding the antigen-binding site of the heavy chain of an antibodycomprising a nucleotide sequence encoding the CDR3 region as shown inFIG. 1 (designated H3).

[0005] The nucleic acid sequence furthermore preferably comprises anucleotide sequence encoding the CDR2 region as shown in FIG. 1(designated H2) and/or a nucleotide sequence encoding the CDR1 region asshown in FIG. 1 (designated H1). More preferably, the nucleotidesequences encoding the CDR3, CDR2 and CDR1 regions are arranged in amanner wherein a polypeptide encoded by the nucleotide sequence iscapable of forming an antigen-binding site having substantially the samecharacteristics as the heavy chain antigen-binding site of themonoclonal antibody G250.

[0006] A further aspect of the present invention relates to a nucleicacid encoding the antigen binding site of the light chain of an antibodycomprising a nucleotide sequence encoding the CDR3 region as shown inFIG. 1 (designated L3).

[0007] Preferably the nucleic acid further comprises a nucleotidesequence encoding the CDR2 region as shown in FIG. 1 (designated L2)and/or a nucleotide sequence encoding the CDR1 region as shown FIG. 1(designated L1).

[0008] More preferably, the nucleic acids encoding the CDR3, CDR2 andCDR1 region are arranged such that a polypeptide encoded by the nucleicacid has substantially the same antigen-binding characteristics as thelight chain antigen binding site of the antibody G250.

[0009] In the nucleic acid of the invention the complement determiningregions CDR3, CDR2 and CDR1 are preferably separated by nucleotidesequence portions encoding so-called framework regions of antibodies.The framework regions may be derived from any species, e.g. from mouse(as shown in FIG. 1 or FIG. 6), it is, however, possible to useframework regions from different species, e.g. human framework regions.It should be noted that also the CDR1, CDR2 and/or CDR3 regions may bemodified, e.g. by modifying the nucleotide sequence resulting in amodified nucleotide sequence encoding a polypeptide sequence differingfrom the polypeptide sequence as depicted in FIG. 1 or FIG. 6, providedthat the antigen-binding specificity remains substantially the same.More preferably, however, the nucleic acid sequences of the heavy chainand light chain CDR3 sequence and of the CDR2 and CDR1 sequence, ifpresent, have the nucleotide sequence as depicted in FIG. 1 or/and thenucleic acid sequences have the nucleotide sequence as depicted in FIG.1.

[0010] Further, the light chain or/and the heavy chain may have theamino acid sequence as depicted in FIG. 6. Thus, the nucleic acid of thepresent invention may comprise a sequence encoding the light chainor/and the heavy chain as shown in FIG. 6.

[0011] The nucleic acid sequences of the present invention may belocated on a recombinant vector comprising at least a copy of a heavychain nucleic acid and/or at least a copy of a light chain nucleic acid.The heavy chain nucleic acid and the light chain nucleic acid arepreferably in operative linkage with an appropriate expression controlsequence, particularly an expression control sequence which isfunctionally in eukaryotic cells. The heavy chain and the light chainnucleic acid may be located on the same vector in operative linkage witha single expression control sequence or with separate expression controlsequences which may be the same or different. Alternatively, the heavychain nucleic acid sequence and the light chain nucleic acid sequencemay be located on different recombinant vectors, each in operativelinkage with a separate expression control sequence.

[0012] Thus, a further aspect of the present invention is a recombinantvector system comprising at least one copy of a nucleic acid encodingthe antigen-binding site of the heavy chain of an antibody comprising anucleotide sequence encoding the CDR3 region (designated H3), or/andencoding the CDR2 region (designated H2), or/and-encoding the CDR1region (designated H1), as shown in FIG. 1 or/and FIG. 6, and at leastone copy of a nucleic acid encoding the antigen-binding site of thelight chain of an antibody comprising a nucleotide sequence encoding theCDR3 region (designated L3), or/and encoding the CDR2 region (designatedL2), or/and encoding the CDR1 region (designated L1), as shown in FIG. 1or/and FIG. 6, wherein the nucleic acid encoding the antigen-bindingsite of the heavy chain and of the light chain have separate expressioncontrol sequences.

[0013] The recombinant vector system comprises a first recombinantvector comprising at least one copy of a nucleic acid encoding theantigen-binding site of the heavy chain and a second recombinant vectorcomprising at least one copy of a nucleic acid encoding theantigen-binding site of the light chain.

[0014] Alternatively, in the recombinant vector system, at least onecopy of the nucleic acid encoding the antigen-binding site of the heavychain and of the light chain are located on the same recombinant vector.

[0015] Further, the present invention comprises a method for therecombinant production of a polypeptide having an antigen-binding sitecomprising:

[0016] (a) providing a nucleic acid as defined above or/and providing arecombinant vector system as defined above,

[0017] (b) introducing the nucleic acid into a suitable host cell,

[0018] (c) culturing the host cell under suitable conditions in a mediumwhereby an expression of the nucleic acid takes place and

[0019] (d) obtaining the expressed product from the medium and/or thehost cell.

[0020] Preferably, the host cell is a eukaryotic cell, particularly amammalian cell. For example, the host cell may be a non-producerhybridoma cell or a CHO cell.

[0021] Between steps (a) and (b) of the method as outlined above amodification of the nucleic acid sequence may take place, wherein themodification substantially does not alter the amino acid sequence of theantigen-binding site of the polypeptide to be expressed. The expressedproduct obtained by the method as outlined above may be used for thepreparation of a diagnostic or therapeutic agent. Thereby it is possibleto couple the antigen-binding polypeptide to a diagnostic marker, e.g. amarker which is useful for in vitro diagnostic methods using a sampleobtained from a patient, e.g. a body fluid or a tissue section, or forquality control. Further, the expressed product may be coupled to adiagnostic marker which is suitable for in vivo applications, e.g. aradioactive marker which is suitable for radioimaging procedures. Fortherapeutical applications the expressed product may be coupled to acytotoxic agent, e.g. a radionuclide, a toxin such as cholera toxin orricin.

[0022] The expressed product which is obtained by the method as outlinedabove is a polypeptide having an antigen-binding site. For example, theexpressed product may be selected from antibodies, e.g. chimerizedantibodies, humanized antibodies, heterobispecific antibodies, singlechain antibodies etc. and from antibody fragments, e.g. antibodyfragments containing an antigen-binding site wherein said antibodyfragments may be obtained by proteolytic digestion of whole antibodiesor by recombinant techniques.

[0023] The manufacture of chimeric antibodies is described e.g. byMorrison et al. (Proc. Natl. Acad. Sci. USA 81 (1984), 6851-6855), whichis herein incorporated by reference. The manufacture of humanizedantibodies is described, e.g. in Jones et al. (Nature 321 (1986),522-525), Riechmann et al. (Nature 332 (1988), 323-329) and Presta(Curr. Opin. Struct. Biol. 2 (1992), 332-339) which are hereinincorporated by reference.

[0024] Single chain antibodies or antibody fragments may be prepared asdescribed in Hoogenboom et al. (Immunol. Rev. 130 (1992), 41-68), BarbasIII (Methods: Companion Methods Enzymol. 2 (1991), 119) and Plückthun(Immunochemistry (1994), Marcel Dekker Inc. Chapter 9, 210-235), whichare herein incorporated by reference.

[0025] Further, the present invention is explained in detail by thefollowing examples:

EXAMPLE 1

[0026] Isolation, cloning and sequencing of the G250 tumor-associatedantigen-specific immunoglobulin variable heavy and light chain domainsfrom the G250 monoclonal antibody producing hybridoma.

[0027] General Strategy

[0028] The variable region genes for the heavy and light chains, whichdetermine the binding specifity of the antibody, were cloned from theG250 murine hybridoma using standard cloning techniques as decribed inMolecular Cloning; A Laboratory Manual (Cold Spring Harbour Press, ColdSpring Harbour, N.Y.) by Maniatis, T. et al.

[0029] The strategy for cloning the variable regions for the heavy andlight chain genes from the G250 hybridoma was achieved by PCRamplification of cDNA obtained from the G250 monoclonal antibodyproducing hybridoma cells.

[0030] Cloning of G250 VH and VL cDNA

[0031] Obtaining the G250 VH and VL chain sequences from the G250monoclonal antibody producing hybridoma was achieved by PCR (Maniatis,T. et al.) amplification of cDNA obtained from the respective clone.

[0032] To obtain cDNA, total RNA was isolated from the G250 producinghybridoma cells according to the method by Chomczynski et al.(Chomczynski, P. and Sacchi, N., Anal. Biochem. 162 (1987), 156-159) andconverted into cDNA essentially as described by Maniatis et al.

[0033] Amplification of cDNA sequences by PCR is possible only, if thesequence of the gene of interest is known. In general, for PCR twoprimers complementary to the 5′-end and the 3′-end of the sequence areused as the initiation point of DNA synthesis. Because the sequence ofthe 5′-ends of the VH and VL chain from the G250 monoclonal antibodyproducing hybridoma cells were unknown, the PCR method, referred to asRACE (rapid amplification of cDNA ends) was used to amplify the VH andVL chain. This was achieved by employing anchor and anchor-poly-Cprimers and the constant VH and VL-primers as shown in FIG. 2. The VHand VL fragments were purified and ligated into pGEM 11 as described byManiatis et al. A ligation mixture was introduced into bacteria, whichwere selected and expanded. DNA was isolated from the selected bacterialcolonies and analyzed by restriction enzyme digestion to confirm thepresence of the amplified VH and VL fragments. Three positive colonieswere subjected to DNA sequencing. The sequences of these threeindividual clones were compared and found to be identical.

[0034] Portions of the resulting sequences including theantigen-specific CDR regions are shown in FIG. 1.

EXAMPLE 2

[0035] Sequencing of cDNA sequences encoding variable heavy and lightchain domains of the G250 monoclonal antibody

[0036] Strategy

[0037] The G250 VH and VL chain cDNA sequences were obtained asdescribed in co-pending U.S. patent application 60/327,008, example 3.The resulting cDNA fragments, a 2.3 kb EcoRI heavy chain variable regionfragment and a 5.5 kb HindIII light chain variable region fragment werecloned into suitable expression vectors which contain the human G1constant region (for the H-chain) or the human Kappa constant region(for the L-chain), respectively, and genes conferring resistance toselectable markers. Competent bacteria (E.coli TG1) were transformedwith the plasmids. Ampicillin resistant clones were selected andexpanded. Plasmid DNA was isolated using the Nucleobond AX 500 MaxiprepKit from Machery & Nagel (Germany). The isolated DNA was subjected tocycle sequencing using the DYEnamic ET Terminator Cycle Sequencing Kit(Amersham Biosciences, Freiburg, Germany) and the resulting DNAmolecules labeled with multiple fluorescent dyes were analyzed using theABI PRISM Model 377 DNA Sequencer (Applied Biosystems, Weiterstadt,Germany). The employed sequencing primers are shown in the following.For sequencing of the full length inserts, the 2.3 kb EcoRI and 5.5 kbHindIII, respectively, a primer-walking approach was applied. Theobtained Sequences of both the CDR's as well as the heavy and lightchain is shown in FIG. 3.

[0038] Primers used for Cycle Sequencing of the variable region of G250heavy (H) and light (L) chain RightH GAG GTT CCT TGA CCC CAG T LeftH CGATTC CCA GTT CCT CAC A RightL AAC GTC CAC GGA TAG TTG CT LeftL CAG AACAGC ATG GGC TTC A

[0039] The sequencing results are shown in FIG. 3. The primer sequencesare underlined. The CDR sequences are boxed.

EXAMPLE 3

[0040] Polypeptide binding specifity and peptide mass fingerprinting

[0041] Binding specifity of the gene product encoded by the sequencesidentified in examples 1 and 2 was tested by means of a sandwich-typeELISA using a G250 anti-idiotypic mouse monoclonal antibody as captureand detection antibody and chimeric G250 antigen for the calibrationcurve. ELISA analysis demonstrated the presence of G250 antibodies (>6μg/ml) in the supernatant of a transfected cell line.

[0042] For protein chemical analysis supernatant from a cell cultureexpressing G250 antibody was collected. The IgG fraction was enrichedusing a one-step protein G-chromatography. An aliquot of the elutedfraction was subjected to SDS-PAGE. A total of five bands with theapparent molecular weight of heavy or light chains were subjected to apeptide mass fingerprint analysis by MALDI mass spectroscopy. Ananalysis of heavy and light chain peptides demonstrated identity of theantibody produced by the cell line with the original mouse G250antibody. The analysis confirmed the presence of peptide fragmentsspecific for CDR1, CDR2, and CDR3 of the heavy chain and CDR2 of thelight chain.

EXAMPLE 4

[0043] Sequence Verification and Mass Spectrometric Characterization ofthe Recombinant Antibody WX-G250

[0044] Introduction

[0045] WX-G250 represents a chimeric antibody (cG250) directed againstthe antigen G250 (carbonic anhydrase 9), a protein expressed on kidneytumor (RCC) cells. G250 is a potential target protein for kidney tumor.The antibody cG250 consists of two identical heavy chains of approx. 50KD and two identical light chains of approx. 25 KD.

[0046] This study was carried out to examine three different aspects ofWX-G250 structure:

[0047] 1) Verification of the amino acid sequence

[0048] 2) Characterization of the configuration of its disulfide bridges

[0049] 3) Examining posttranslational modifications like glycosylations

[0050] Abbreviations

[0051] KD kilo Dalton

[0052] MALDI Matrix-assisted laser desorption ionization

[0053] MS Mass spectrometry

[0054] TOF Time of flight

[0055] PMF Peptide mass fingerprint

[0056] SDS Sodium dodecylsulfate

[0057] PAGE Polyacrylamide gel electrophoresis

[0058] MW Molecular weight

[0059] DTT Dithiothreitol

[0060] MH+ single charged protonated peptide mass

[0061] exp experimental

[0062] th theoretical

[0063] RP Reversed Phase

[0064] HPLC High Performance Liquid Chromatography

[0065] hc Heavy Chain

[0066] Ic Light Chain

[0067] Materials and Methods:

[0068] 1. Materials

[0069] 1.1. Desalting: C18 ZipTip, C4 ZipTip, Millipore, Bedford, Mass.,USA.

[0070] 1.2. MALDI mass spectrometry: Voyager STR, Applied Biosystems,Foster City, Calif., USA

[0071] 1.3. Nano-ESI mass spectrometry: QSTAR Hybrid Quadrupole-TOFLC/MS/MS (022222-44) Perkin-Elmer Sciex Instruments, Foster City,Calif., USA

[0072] 1.4. HPLC of enzymatic digests: HP 1100 (Agilent)

[0073] 1.5. HPLC of reduced and alkylated antibody: HP 1090 (Agilent)

[0074] 1.6. LC-MS/MS: Ultimate nano-LC system (Dionex), EsquireLC iontrap mass spectrometer (Bruker Daltonics)

[0075] 1.7. Proteases: Trypsin, Lys-C, Glu-C, Asp-N, Roche Diagnostics,Basel, Switzerland

[0076] 1.8. N-Glycosidase F: Roche Diagnostics, Basel, Switzerland

[0077] 1.9. SDS-PAGE: 12% Polyacrylamide minigel (BioRad), Gel chamber(BioRad), Laemmli buffer, Coomassie Blue G250 (Sigma), MW-Marker(Invitrogen)

[0078] 1.10. All used chemicals were from Sigma Aldrich, Munich, Germany

[0079] 2. Methods

[0080] Mass Spectrometric Analysis of Intact WX-G250 (MALDI-MS Analysis)

[0081] The sample was desalted using a C4-ZipTip (Millipore according tothe manufacturers protocol. The eluted sample was mixed 1:1 with thematrix solution sinapinic acid and analyzed by MALDI-MS. The massspectrometer (Voyager STR, Applied Biosystems) was calibrated externallywith an IgG standard (Sequazyme™ Peptide Mass Standard Kits, AppliedBiosystems).

[0082] Mass Spectrometric Analysis of Light and Heavy Chains afterReduction and Alkylation of Intact WX-G250 (MALDI-MS, ESI-MS)

[0083] The sample was reduced with dithiothreitol (DTT, 7 μg/μl) for 20minutes at 55° C. and alkylated with iodacetamide (IAA, 18,6 μg/μl) for20 minutes at room temperature. The sample was desalted by a C4-ZipTip(Millipore) according to the manufacturers protocol. The eluted samplewas mixed 1:1 with matrix solution sinapinic acid and analyzed byMALDI-MS. The mass spectrometer (Voyager STR, Applied Biosystems) wascalibrated externally with a protein standard mixture (Sequazyme™Peptide Mass Standard Kits, Applied Biosystems).

[0084] For ESI-MS measurements a QStar (Sciex-Applied Biosystems)equipped with a nanospray source (Protana) was used. The QStar is a QTOFinstrument using a TOF as ion mass discriminator. The desalted andlyophilized sample was dissolved in 50% acetonitrile, 0.5% formic acid(v/v). 2 μl of the sample was centrifuged into a nanospray needle(Protana). The needle was then built in into the nanospray source andconnected with an electrode. The needle was broken very carefully toallow a homogenous ion spray. The voltage was increased until acontinuous nanospray was reached.

[0085] Light and Heavy chains of 10 μg of the reduced and alkylatedWX-G250 antibody were separated by reversed phase HPLC. A HP1090 HPLC,and a RP C4 column (Vydac Protein C4, 2 mm×250 mm) was used for thisseparation. The gradient conditions were the following: solvent A: 0.1%TFA in water, solvent B: 0.1% TFA in 80% acetonitrile; gradient: 0 min.30% B, 5 min. 30% B, 60 min. 75% B, 75 min. 95% B, 85 min. 95% B, 90min. 30% B. The flow was 1 ml/min.

[0086] The fractions were subsequently analyzed by SDS-PAGE.

[0087] Mass Spectrometric Analysis of Proteolytic Digests of WX-G250with Trypsin, LysC, AspN and GluC by MALDI Peptide Mass Fingerprinting(PMF)

[0088] The endoprotease trypsin cleaves specifically C-terminal of thebasic amino acid residues lysine (K) and arginine (R). The endoproteaseLysC cleaves specifically C-terminal of the amino acid residue lysine(k). AspN cleaves specifically N-terminal of the amino acid residueaspartic acid (D). GluC cleaves specifically C-terminal of the aminoacid residue glutamic acid (E). Cyanogen bromide (BrCN) cleavesspecifically C-terminal of the amino acid residue methionine. Forcyanogen bromide cleavage of the intact antibody WX-G250 was incubatedovernight at room temperature in 70% formic acid added with 100 mM BrCN.

[0089] The resulting peptide mixtures after digest representcharacteristic fingerprints for each protein, depending on thecorresponding protein sequence.

[0090] For the digest with the enzymes trypsin, LysC, and AspN the samedigestion protocol was used: WX-G250 was digested over night at 37° C.(c: 1 μg/μl in 2 M urea, 400 mM NH4HCO3) after reduction (45 mMDithiothreol (DTT) in 8 M urea, 400 mM NH4HCO3, 30 min, 50° C.) andalkylation (100 mM lodacetamid in 8 M urea, 400 mM NH4HCO3, 30 min 50°C. at room temperature). For GluC the only difference to the protocoldescribed above was that the incubation was done at room temperature.The samples were desalted with a C18-ZipTip (Millipore) according to themanufacturers protocol. The eluted peptides were mixed 1:1 with matrixsolution (2,5-dihydroxybenzoic acid (DHB): 2-hydroxy-5-methoxybenzoicacid 9:1) and analyzed by MALDI-MS. The resulting peptide masses werecompared with the respective tryptic in-silico digests using the MSdigest program of Protein Prospector V3.2.1. For the in-silico digesttwo miscleavages (lysine or arginine where trypsin has not cleaved) wereallowed. For the MALDI-TOF-MS measurements a Voyager STR (AppliedBiosystems) was used. The used mass range of the MALDI-TOF-MS analysiswas from 700-4200 Da. The autotryptic masses of 805.41 Da and 2163.05 Dawere used for internal calibration. After internal calibration the massaccuracy was better than 50 ppm.

[0091] MALDI PMF without Reduction after Cleavage of WX-cG250 withTrypsin, LysC, AspN, GluC, and BrCN

[0092] For the determination of disulfide bridges the complete cG250(Heavy and Light chain) was digested without prior reduction andalkylation.

[0093] Detection of other Posttranslational Modifications by MALDI MS ofHPLC-separated Peptides (Trypsin, AspN, LysC, and GluC Digests)

[0094] Due to suppression effects in MALDI MS especially glycopeptidesoften are not detectable in a complex mixture. Due to the low massdifference of 1D and the overlapping isotope patterns of peptidesdiffering in 1D, deamidated peptides cannot be detected in presence ofnon-deamidated peptides. Therefore, the peptides were separated by HPLC.

[0095] Digestion was performed according to the manufacturers protocol.WX-G250 (c: 0.5 μg/μl) was denatured in 1% SDS, 100 mM PBS, pH 7.3, 1%mercaptoethanol and diluted in 0.1% SDS, 1% CHAPS, 100 mM PBS, pH 7.3,1% mercaptoethanol (c: 0.05 μg/μl) WX-G250 was incubated with five unitsof N-Glycosidase F overnight. The solution was delivered to Wilex forisoelectric focussing.

[0096] Peptide mixtures were separated on a 300 μm×150 mm capillary HPLCcolumn using a linear acetonitrile gradient with a slope of 0.57% B/minstarting from 2% B to 45% B in 75 minutes. Solvent A: was 5%acetonitrile, 0.1% trifluoracetic acid, solvent B 80% acetonitrile, 0.1%trifluoracetic acid, the column was from LC-Packings, filled with VydacRP18, 5 μ, 300 Å material. The HPLC system used was a HP 1100 systemfrom Agilent.

[0097] LC-MS and LC-MS/MS of Tryptic Digest of cG250

[0098] The tryptic peptide mixture of cG250 was separated using a 75μm×150 mm capillary HPLC column (RP18, Dionex) at a flow rate of 200 nl.MS was performed with a quadrupole ion trap (Esquire, Bruker Daltonics).The two most intensive signals of each spectrum were fragmented (MS/MS).

[0099] SDS-PAGE

[0100] To evaluate the efficiency of the separation of the Light andHeavy chains by RP-HPLC the HPLC fractions were applied to a 12%SDS-PAGE gel. 50% of the HPLC fractions were dried in a vacuumconcentrator and subsequently solved in SDS-PAGE sample loading buffer(25 mM Tris/HCI, pH 7.5, 2% SDS, 1% DTT, 15% glycerole). As MW standarda protein standard from Invitrogen was used (10-220 KD). The gel was runat 150 V for 1.5 hours according to Laemmli et al. Then it was stainedwith Coomassie Blue G250 for 1 hour.

[0101] Edman Sequencing of Selected HPLC Fractions after EnzymaticDigest

[0102] To verify the posttranslational modifications of the trypsindigest and fractions of the LysC digest were analyzed by automated Edmansequencing.

[0103] Results:

[0104] Mass Spectrometric Analysis of Intact WX-G250 (MALDI-MS Analysis)

[0105] The linear mode MALDI-MS spectrum showed signals of the single totriple charged ions of the intact antibody (MWexp.: 149135 D, MWth.:147424 D). The mass spectrometer was externally calibrated with anantibody standard (Applied Biosystems). The difference between thetheoretical and the experimentally determined MW might result fromglycosylation. The mass accuracy in this MW range is approximately100-150 ppm.

[0106] Mass Spectrometric Analysis of Light and Heavy Chains afterReduction and Alkylation of Intact WX-G250 (MALDI-MS, ESI-MS)

[0107] To detect the masses of the separated Light and Heavy chains ofWX-G250 the antibody was reduced and alkylated as described in Materialsand Methods, desalted by ZipTip (Millipore), and then applied to themass spectrometer.

[0108] The MALDI-MS spectrum shows signals of the single and doublecharged ions of the Light chain. (MWexp.: 23886 D, MWth.: 23873 D) and asignal of a protein (Heavy chain) at m/z: 51507 D (MWth,: 49839 D). TheMW of the Light chain represents the theoretically expected mass(difference: 13 D), whereas between the theoretical and experimental MWof the Heavy chain a significant difference of 1.668 D was observed.This finding leads to the assumption that the antibody is glycosylatedonly at its Heavy chain.

[0109] Due to an improved resolution compared to MALDI-MS threedifferent isoforms of WX-G250 were detected. The ESI-MS spectrum showedstrong signals of the 15-fold to 34-fold charged ions of the light chainand very weak signals of the 53-fold to 57-fold charged ions of threedifferent proteins at about 52 KD (light chain: 23869 D, other proteins:51036 D, 51201 D, 51328 D; theoretical masses: Ic: 23873, hc: 49839).The mass difference between the three Heavy chain isoforms found was 165D and 128 D. The latter mass difference corresponds to a lysine residue(128 D). The accuracy of the ESI-MS was better than 50 ppm.

[0110] 10 μg of the reduced and alkylated antibody was separated byreversed phase HPLC. The fractions were analyzed by SDS PAGE andMALDI-TOF MS. Fractions 4 and 5 contained light and heavy chain ofWX-G250.

[0111] Mass Spectrometric Analysis of Proteolytic Digests of WX-G250with Trypsin, LysC, AspN and GluC by MALDI Peptide Mass Fingerprinting(PMF)

[0112] The digests of the whole antibody were performed to confirm theamino acid sequence.

[0113] Peptides derived from Light and Heavy chains of WX-G250 from thePMFs digested with different enzymes were found.

[0114] Some of the peptides of the tryptic and LysC digests containeslysine or arginine residues. These miscleavages are not unusual andoccur probably due to lack of enzyme access at certain cleavagepositions.

[0115] To check if the C-terminal part of the heavy chain was somehowmodified, the whole antibody was incubated with BrCN in 70% formic acidovernight at room temperature. For this digest cG250 was not reduced andalkylated. One peptide derived from the Light chain was found as well.Some other peptides (MH+: 816.41 D, 871.40 D, 877.40 D, 887.41 D,1277.65 D, 1305.60 D, 1333.64 D) were found as well. However, thesepeptides could not be explained by the theoretical digest of WX-G250.

[0116] Probably these peptides were due to chemical modifications asobtained by the homoserine lactone from the Light chain.

[0117] MALDI PMF without Reduction after Cleavage of WX-cG250 withTrypsin, LysC, AspN, GluC, and BrCN

[0118] The disulfide bridges found in the complete cG250 (Heavy andLight chain) are summarized in Table 1. A schematic summary is given inFIG. 4.

[0119] The mass difference between the carbamidomethylated cysteins (seereduced and alkylated peptides) and the unmodified cysteine residues is57 D.

[0120] Table 1: Disulfide Bridges in WX-cG250 Light Chain: 3559.1127-142/191-207 Cys134-Cys194 linear mode tryptic digest 3824.4127-142/189-207 Cys134-Cys194 linear mode tryptic digest 5256.8 19-24/62-103 Cys23-Cys88 linear mode tryptic digest 6046.6 19-24/55-103 Cys23-Cys88 linear mode tryptic digest 6251.9 10-24/62-103 Cys23-Cys88 linear mode tryptic digest 3559.0127-142/191-207 Cys134-Cys194 reflector mode tryptic digest 3824.4127-142/189-207 Cys134-Cys194 reflector mode tryptic digest 3887.4127-145/191-207 Cys134-Cys194 reflector mode LysC digest 4152.7127-145/189-207 Cys134-Cys194 reflector mode LysC digest Heavy Chain:3464.1  20-38/88-98 Cys22-Cys96 linear mode tryptic digest 4744.4 20-38/77-98 Cys22-Cys96 linear mode tryptic digest 2311.1258-276/323-324 Cys263-Cys323 LC-MS/MS tryptic digest Intermoleculardisulfide bridge: 1842.8 208-214 (Ic)/ Cys214-Cys222 reflector mode LysC216-224 (hc) digest

[0121] Detection of other Posttranslational Modifications by MALDI MS ofHPLC-separated Peptides (Trypsin, AspN, LysC, and GluC Digests)

[0122] One N-glycosylation site was characterized at N 299 (hc). Thesequence showed the NST motif, which represents a potentialN-glycosylation site. According to the data required by MALDI-MS massthree different variants of complex type N-glycosylation (4×GlcNAc, 3-5Hexose, 1×Fucose) were found. The three isoforms differed by one and twohexoses, respectively (mass difference: 162 D).

[0123] Two deamidation sites were located. Asparagine was deamidated toaspartic acid. One of these sites was located in the Light chain (aminoacid residues N 137 or N 138). Due to the two neighbored asparagineresidues it could not be detected which of them was deamidated.

[0124] Another deamidation site was found in Heavy chain, position N317. It was detected by MALDI-MS in a tryptic peptide after HPLCfractionation.

[0125] From the peptide mass fingerprints which comprised about 99% ofthe WX-G250 sequence no O-linked glycosylation sites were detected.

[0126] A summary of the results can be taken from FIG. 5.

[0127] LC-MS and LC-MS/MS of Tryptic Digest of cG250

[0128]FIG. 6 shows the sequence coverage of WX-G250 in the LC-MS/MSexperiment of a tryptic digest without reduction and alkylation of theantibody.

[0129] The underlined sequences were detected as tryptic peptides.Detected disulfide bonds are marked. Cysteine residues undetected inthis experiment are bold and underlined.

[0130] Edman Sequencing

[0131] To verify the posttranslational modifications fractions 5 and 33of the trypsin digest and fractions 17 and 21 of the LysC digest wereanalyzed by automated Edman sequencing.

[0132] Tryptic fraction 5 contained the expected sequence E E Q Y ?corresponding to residues 295-298 (hc). The glycosylated N following theY cannot be seen in Edman sequencing. Together with the peptide mass forpeptide 295-303 determined by MALDI-MS it could be proven that thissequence was indeed glycosylated at position 300. Two minorcontaminations were also found in this HPLC fraction: VSITC* and LIVSL.

[0133] VSITC* was derived from a Light chain peptide starting atposition 19. It contained a Cys modified by iodacetamide. LIVSL couldnot be annotated to the WX-G250 structure. It is possible that thispeptide was derived from trypsin.

[0134] Tryptic fraction 33 was not successful. No sequence could bedetermined, probably due to the limitation in sample amount (>>1 pmol).

[0135] LysC fraction 17 was close to the detection limit (<0.5 pmol) butproved to be the expected sequence: S? G? T? A S V V? C? L L?. However,due to limited amount of sample it was not possible to sequence to theexpected deamidation site which followed the two leucins. But togetherwith the MALDI-MS data the deamidation is evident.

[0136] LysC fraction 21 clearly showed the expected sequence T K P R Ecorresponding to residues 291-295 (hc). Together with the peptide massfor peptide 291-319 determined by MALDI-MS it could be proven that thissequence was indeed glycosylated at position 300. This is in accordancewith the Edman sequencing result of tryptic fraction 5.

[0137] Fraction 23 of Lys C digest was analyzed by Edman sequencing toprove the identity of this very long fragment 150-202 (hc) which couldonly be detected in linear mode MALDI-MS. However, due to the smallamount of sample it was impossible to get sequence information from thissample.

[0138] Conclusion

[0139] Sequence Verification

[0140] 86% of the sequence of G250 was covered by peptide massfingerprints using four different enzymes: trypsin, LysC, AspN, andGluC. Additional measurements in MALDI-MS linear mode increased thesequence coverage up to 99%. No deviations or mutations from thetheoretically expected sequence were determined. However, one sequenceheterogeneity (C-terminal lysine) was identified by peptide massfingerprinting in reflector mode.

[0141] Verification of the Configuration of Disulfide Bonds

[0142] After peptide mass fingerprinting measured in reflector mode andlinear mode MALDI mass spectrometry, respectively, four disulfidebridges (out of ten) were clearly determined. In Light chain disulfidebridges between Cys23-Cys88 and Cys-134-Cys194 were detected. In Heavychain a disulfide bridge between Cys22-Cys96 was detected. Further, onedisulfide bond connecting one Light and one Heavy chain could beidentified: it was located at Cys214 (lc)-Cys222 (hc).

[0143] A summary of these results is shown in FIG. 4.

1 23 1 23 DNA Artificial oligonucleotide primer that amplifies mouseanti-human monoclonal antibody cDNA 1 gcatgcgcgc ggccgcggag gcc 23 2 35DNA Artificial oligonucleotide primer that amplifies mouse anti-humanmonoclonal antibody cDNA 2 gcatgcgcgc ggccgcggag gccccccccc ccccc 35 348 DNA Artificial oligonucleotide primer that amplifies mouse anti-humanmonoclonal antibody cDNA 3 ctctaagctt ggctcaaaca cagcgacctc ggatacagttggtgcagc 48 4 45 DNA Artificial oligonucleotide primer that amplifiesmouse anti-human monoclonal antibody cDNA 4 ctcttctaga gagtctctcagctggtagga tacagttggt gcagc 45 5 357 DNA Artificial mouse anti-humanmonoclonal antibody cDNA 5 gacgtgaagc tcgtggagtc tgggggaggc ttagtgaagcttggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt aactattacatgtcttgggt tcgccagact 120 ccagagaaga ggctggagtt ggtcgcagcc attaatagtgatggtggtat cacctactat 180 ctagacactg tgaagggccg attcaccatt tcaagagacaatgccaagaa caccctgtac 240 ctgcaaatga gcagtctgaa gtctgaggac acagccttgttttactgtgc aagacaccgc 300 tcgggctact tttctatgga ctactggggt caaggaacctcagtcaccgt ctcctca 357 6 119 PRT Artificial mouse anti-human monoclonalantibody 6 Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Leu GlyGly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe SerAsn Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu GluLeu Val 35 40 45 Ala Ala Ile Asn Ser Asp Gly Gly Ile Thr Tyr Tyr Leu AspThr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn ThrLeu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala LeuPhe Tyr Cys 85 90 95 Ala Arg His Arg Ser Gly Tyr Phe Ser Met Asp Tyr TrpGly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 7 321 DNAArtificial mouse anti-human monoclonal antibody cDNA 7 gacattgtgatgacccagtc tcaaagattc atgtccacaa cagtaggaga cagggtcagc 60 atcacctgcaaggccagtca gaatgtggtt tctgctgttg cctggtatca acagaaacca 120 ggacaatctcctaaactact gatttactca gcatccaatc ggtacactgg agtccctgat 180 cgcttcacaggcagtggatc tgggacagat ttcactctca ccattagcaa tatgcagtct 240 gaagacctggctgatttttt ctgtcaacaa tatagcaact atccgtggac gttcggtgga 300 ggcaccaagctggaaatcaa a 321 8 107 PRT Artificial mouse anti-human monoclonalantibody 8 Asp Ile Val Met Thr Gln Ser Gln Arg Phe Met Ser Thr Thr ValGly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val ValSer Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys LeuLeu Ile 35 40 45 Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg PheThr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn MetGln Ser 65 70 75 80 Glu Asp Leu Ala Asp Phe Phe Cys Gln Gln Tyr Ser AsnTyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 1059 2431 DNA Artificial mouse anti-human monoclonal antibody cDNA 9tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctt caagaattct 60tcagatacaa agaatctcta aaccctgagg acattctatc acaaataagt aaaattcaga 120aaattctgaa tgctcccatc acagagatga atctgctatg aacagctcat aggtgtgaag 180ctctacaaaa gccatattat tgaaaagcca cattgtgccc agactttgga aagactgagc 240tcatatcctg aaatacagtt atgtgtggtt ctatctaatt acacatttac actaaggaaa 300catggcagta tgggaatgaa gcttgttctg tacacattaa cagagggaaa ctaaacaaag 360tatggtgaat ccctaaccaa aagtaaaaaa aaaaaaaaaa aagaaaagaa aagaaaaaaa 420aagtgaaact acaatatgtt tcaaatgctg taactgaaat ctggtttttt gatgccttat 480atctgttatc atcagtgact tcagatttag tccaactcca gagcatggta tagcaggaag 540acatgcaaat aggtcttctc tgtgcccatg aaaaacacct cggccctgac cctgcagctc 600tgacagagga ggcctgtcct ggattcgatt cccagttcct cacattcagt gatcagcact 660gaacacagac ccctcaccat gaacttcggg ctcagattga ttttccttgt cctggtttta 720aaaggtatct tattgagtat agaggacatc tgctgtatgc acagaggtgc agaaaaaatg 780ttgtttgttt tttttagtga caatgctcca aacagtattc tttctttgca ggtgtcctgt 840gtgacgtgaa gctcgtggag tctgggggag gcttagtgaa gcttggaggg tccctgaaac 900tctcctgtgc agcctctgga ttcactttca gtaactatta catgtcttgg gttcgccaga 960ctccagagaa gaggctggag ttggtcgcag ccattaatag tgatggtggt atcacctact 1020atctagacac tgtgaagggc cgattcacca tttcaagaga caatgccaag aacaccctgt 1080acctgcaaat gagcagtctg aagtctgagg acacagcctt gttttactgt gcaagacacc 1140gctcgggcta cttttctatg gactactggg gtcaaggaac ctcagtcacc gtctcctcag 1200gtaagaatgg cctctccagg tctttttttt aatctttgta atggagtttt ctgaacattg 1260cagactaatc ttggatattt gtccctgagg tagccggctg agagaaattg ggaattaaac 1320tgtctcggga tctcagagcc tttaggacag attatctcca catctttgaa aaactgagat 1380tctgtgtgat ggtgttggtg gagtccctgg atgatgggat agggactttg gaggctcatt 1440tgagggagat gctaaaacaa tcctatggct ggagggagag ttggggctgt agttggagat 1500tttcagtttt tagaataaaa gctttagctg cgggaaatcc ttcaggacca cctctgtgac 1560agcatttata cagtatccga tgcataggga caaagagtgg agtggggcac tttctttcga 1620tttgtgggga atgttccaca ctagtttctg tgaaacctca tttgttggag ggagagctgt 1680cttagtgcct gagtcaaggg agaagggcat ctagcctcgg tctcaaaagg gtagttgctg 1740tccagagagg tctggtggag cctgcaaaag tccagctttc aaaggaacac agaagtatgt 1800gtatggaata atagaagatg ttgcttttac tcttaagttg gttcatagga aaaatagtta 1860aaactgtgag tttaaaatgt gagagggttt tcaagtactc atttttttac atgtccaaaa 1920tttctgtcaa tcaatttgag gtcttgtttg tgtagaactg acattactta aagtttaacc 1980gaggaatggg agtgaggctc tctcataccc tattcagaac tgacttttaa caataataaa 2040ttaagtttaa aatattttta aatgaattga gcaatgttga gttggagtca agatggccga 2100tcagaaccag aacacctgca gcagctggca ggaagcaggt catgtggcaa ggctatttgg 2160ggaagggaaa ataaaaccac taggtaaact tgtagctgtg gtttgaagaa gtggttttga 2220aacactctgt ccagccccac caaaccgaaa gtccaggctg agcaaaacac cacctgggta 2280atttgcattt ctaaaataag ttgaggattc agccgaaact ggagaggtcc tcttttaact 2340tattgagttc aaccttttaa ttttagcttg agtagttcta gtttccccaa acttaagttt 2400atcgacttct aaaatgtatt tagaattcat t 2431 10 5557 DNA Artificial mouseanti-human monoclonal antibody cDNA 10 aattccaagc tttgtatctt cagatccaggaaagccacca ccaatatcaa acagatacat 60 gctgaaacca acttctgttc ttatgtcaaatgcacagcgg gcatctgaca ctgcctgcat 120 gaaggtctca ggtcaatact tccactacacacatggaagc tgacaccaat gacgtcaata 180 tttagctctt ttgcccattt caggaggagactgctggttt tgagtgtggc accagactta 240 acaccaagtc gacaaactgc tttggaatcatctgtgacaa tccacaaaaa caactttgtc 300 ttacaatgtg ctctgacgac attcatcaattcatttcact gtcaaaagtc atcatctgga 360 ctccattact ggcagcatac ttgatttgagacacttgttt acaaaaatgt gcataggtaa 420 tcctctctgg aggaaccaga agcccccgttccaactgtat ttcagtcttg cttgcacagt 480 caaatcctgt accaatagca gctagggtgttaactatggc tctgttgtcc ttacacttga 540 ctgcacaaaa aggaataaca ttcggaagagcttttagcca cctcagatgc ttctttagaa 600 tgtctctgag gtccggaacc tagaaagaagagacttcatt tattattttg tgttcagaat 660 gtccttagca ctaaagccac catctatgatacagcagtca aactcttcct tagtatagct 720 gctcatcgtt ctccatgtgc ctacagaaaacctagacatg gaattaaatt attgccagcc 780 ccttacaagg tcaacttatc caagaactgtgaatgcagac tccttgaaat gttggaaaca 840 ctcacagcac agggtcaaga ctggctggacacatggagac actgaatcct gaagagcact 900 tagctgtctg ttgcttcatc atgtctactgacctgaggtg gcaccaagct gcttactgag 960 ggaggactgt ggcggtgtct gcaggaactgacaattctcc acaattctct tactgcccca 1020 ctcataactc ttctcttctc catcttcttctttctttcct ctcccctcct ttttcccttt 1080 cactactttt ttcctttctt cttttccacttcccttttct ttcttctttt gctgttgctg 1140 ttgtaaagga tttattgttt cctcgtgattgaaccaaagg tagttgtact attatttctg 1200 taaaactcat ctgttgattt tctattaattaattaatttt gtttacactc catattttat 1260 tcaacccctc catcctccta ctggtctacataccatacct ccttcccaca cccctgtctc 1320 cacatggatg ctgccacctc ccatgccacctgacctctca tctccctagg gcatctagtc 1380 tcttgaggct tagatgcatc atttctgagtgaacacagat ccaacaatcc tctgctatat 1440 gtgtgttggt ggcctcatag cagctggtgtatgctgcctg tttgttgatc cagtgtttga 1500 gaggtctcgc gggttcagat taattgagattgttggacct cctcagcgtc tttcagtctt 1560 tccctgattc aacaacaggg ttcattgtttctgttcattg gttgggtgca aatatctgca 1620 tctgactcag ctgcttattg ggtcttctggagtgcagtca tgctaggtcc gtttctatga 1680 gtgctccata gcctcagtga tagtgtcaggcgttgggact gccccttgac ctggattcta 1740 ttttggacct gtcgctggac cttcttttcctcaggctccc ctccatctgt atccctgtaa 1800 ttctttcaga caggaacaaa tatgggtcagagttgtgagt gtggaatggc acccccttcc 1860 ctcatttaat gccctgtctt cctggtggaagtgggctcta taagttccca ctccctactg 1920 ttgggcattt catccctttg agtcctgagagtctctcacc tcccaggtct ctggtgcatt 1980 ctggagggtc ctcccaacct cctacctccccaggttgcct gttgacagac ttctgctggc 2040 ccccagtgct tcagtccttt tccctcacccaatatctgat ttggatggaa gcctgtcatg 2100 agaacatcta tatacttgtg gtttcagagctttaaattgg tccttgagct tctattttga 2160 gttcctttcc agtgattact tgctgtctttggtagtactt ttgactgttt atttaacctg 2220 gatactctca tacagctgtg taatttacttccttatttga tgactgcttt gcatagatcc 2280 ctagaggcca gcccagctgc ccatgatttataaaccaggt ctttgcagtg agatctgaaa 2340 tacatcagaa cagcatgggc ttcaagatggagtttcatac tcaggtcttt gtattcgtgt 2400 ttctctggtt gtctggtgag aattttaaaagtattataac atctcaaaag taatttattt 2460 aaatagcttt tcctatagga agccaatattaggcagacaa tgccattaga taagacattt 2520 tggattctaa catttgtgtc aaaaatctttgtatatataa gtgtttactc attatctatt 2580 tctgattgca ggtgttgatg gagacattgtgatgacccag tctcaaagat tcatgtccac 2640 aacagtagga gacagggtca gcatcacctgcaaggccagt cagaatgtgg tttctgctgt 2700 tgcctggtat caacagaaac caggacaatctcctaaacta ctgatttact cagcatccaa 2760 tcggtacact ggagtccctg atcgcttcacaggcagtgga tctgggacag atttcactct 2820 caccattagc aatatgcagt ctgaagacctggctgatttt ttctgtcaac aatatagcaa 2880 ctatccgtgg acgttcggtg gaggcaccaagctggaaatc aaacgtaaat agaatccaaa 2940 ctctctttct tccgttgtct atgtctgtggcttctatgtc taaaaatgat gtagatattt 3000 tttctctgag accagattct gtcactctccaaggcaaaga tacatagtca ctccgtaagc 3060 agagctggga ataggctaga catgttctctggagaatgaa tgccagtgta ataattaaca 3120 caagtgatag tttcagaaat gctcaaagaagcagggtagc ctgccctaga caaaccttta 3180 cttggtgctc agaccatgct cagtttttgtatgggggttg agtgaaggga caccagtgtg 3240 tgtatacgtt cggagggggg accaagctggaaataaaacg taagttgtct tctcaactct 3300 tgttcactga gtctaacctt gttactttgttctttgttgt gtgtttttct taaggagatt 3360 tcagggatgt atcaaattcc attctcagatcaggtgttaa ggagggaaaa cttgtcccac 3420 aagaggttgg aatgattttc aggctaaattttaggcttct aaaccaaagt cattaaacta 3480 ggggaagagg gataattgtc tgcctagggagggttttgtg gaagtacagt taaagtagat 3540 cactgtaaac cacattcaga gatgggaccagactggaaat aaaacctaag aacatttttg 3600 ctcaactgct tgtgaagttt tggtcccattgtgtcctttg tgtgagtttg tggtgttcat 3660 tagataaatg aactattcct tgtaacccaaaacttaaata gacgagaacc aaaaatctag 3720 ctactgtata agttgagcaa acagactgacctcatgtcag atttgtggga gaaatgagaa 3780 aggaacagtt tttctctgaa cttggcctatctaactggat cagcctcagg caggtttttg 3840 taaagggggg cacagtgata tgaatcactgtgattcacgt tcggctcggg gacaaagttg 3900 gaaataaaac gtaagtagat ttttgctcatttacttgtga cgttttggtt ctgtttgggt 3960 aactcgtgtg aatttgtgac attttggctaaatgagccat tcctggcaac ctgtgcatca 4020 atagaagatc ccccagaaaa gagtcagtgtgaaagctgag cgaaaaactc gtcttaggct 4080 tctgagacca gttttgtaag gggaatgtagaagaaagagc tgggcttttc ctctgaattt 4140 ggcccatcta gttggactgg cttcacaggcaggtttttgt agagaggggc atgtcatagt 4200 cctcactgtg gctcacgttc ggtgctgggaccaagctgga gctgaaacgt aagtacactt 4260 ttctcatctt tttttatgtg taagacacaggttttcatgt taggagttaa agtcagttca 4320 gaaaatcttg agaaaatgga gagggctcattatcagttga cgtggcatac agtgtcagat 4380 tttctgttta tcaagctagt gagattaggggcaaaaagag gctttagttg agaggaaagt 4440 aattaatact atggtcacca tccaagagattggaccggag aataagcatg agtagttatt 4500 gagatctggg tctgactgca ggtagcgtggtcttctagac gtttaagtgg gagatttggg 4560 ggggatgagg aatgaaggaa cttcaggatagaaaaggtct gaagtcaagt tcagctccta 4620 aaatggatgt gggagcaaac tttgaagataaactgaatga cccagaggat gaaacagtgc 4680 agatcaaaga ggggcctgga gctctgagaacagaaggaga gtcattcgtg ttgagtttcc 4740 acaaatactg tcttgagttt tgcaataaaagtgggatagc agagttgagt gagccatagg 4800 ctgagttctc tcttttgtct cctaagtttttatgactaca aaaatcagta gtatgtcctg 4860 aaataatcat taaactgttt gaaagtatgactgcttgcca tgtagatacc atggcttgct 4920 gaataatcag aagaggtgtg actcttattctaaaatttgt cacaaaatgt caaaatgaga 4980 gactctgtag gaacgagtcc ttgacagacagctcaagggg tttttttcct ttgtctcatt 5040 tctacatgaa agtaaatttg aaatgatcttttttattata atagtagaaa tacagttggg 5100 tttgaactat atgttttaat ggccacggttttgtaagaca tttggccctt tgttttccca 5160 gttattactc gcttgtaatt ttatatcgccagcaatggac tgaaacggtc cgcaacctct 5220 tctttacaac tgggtgacct cgcggctgtgccagccattt ggcgttcacc ttgccgctaa 5280 gggccgtgtg aacccccgag gtagcatcccttgctccgcg tggaccactt tcctgaggca 5340 cagtgatagg aacagagcca ctaatctgaagagaacagag atgtgacaga ctacactaat 5400 gttagaaaaa caaggaaagg gtgacttattggagatttca gaaataaaat gcatttatta 5460 ttatattccc ttattttaat tttctattagggaattagaa agggcataaa ctgctttatc 5520 cagtgttata ttaaaagctt tttttttttcagtgcta 5557 11 19 DNA Artificial oligonucleotide primer that amplifiesmouse anti-human monoclonal antibody cDNA 11 gaggttcctt gaccccagt 19 1219 DNA Artificial oligonucleotide primer that amplifies mouse anti-humanmonoclonal antibody cDNA 12 cgattcccag ttcctcaca 19 13 20 DNA Artificialoligonucleotide primer that amplifies mouse anti-human monoclonalantibody cDNA 13 aacgtccacg gatagttgct 20 14 19 DNA Artificialoligonucleotide primer that amplifies mouse anti-human monoclonalantibody cDNA 14 cagaacagca tgggcttca 19 15 214 PRT Artificial mouseanti-human monoclonal antibody 15 Asp Ile Val Met Thr Gln Ser Gln ArgPhe Met Ser Thr Thr Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys LysAla Ser Gln Asn Val Val Ser Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys ProGly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Asn Arg Tyr ThrGly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe ThrLeu Thr Ile Ser Asn Met Gln Ser 65 70 75 80 Glu Asp Leu Ala Asp Phe PheCys Gln Gln Tyr Ser Asn Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr LysLeu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile PhePro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val ValCys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln TrpLys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu SerVal Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 SerThr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200205 Phe Asn Arg Gly Glu Cys 210 16 449 PRT Artificial mouse anti-humanmonoclonal antibody 16 Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu ValLys Leu Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly PheThr Phe Ser Asn Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Thr Pro Glu LysArg Leu Glu Leu Val 35 40 45 Ala Ala Ile Asn Ser Asp Gly Gly Ile Thr TyrTyr Leu Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn AlaLys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu AspThr Ala Leu Phe Tyr Cys 85 90 95 Ala Arg His Arg Ser Gly Tyr Phe Ser MetAsp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser Ala SerThr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys SerThr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp TyrPhe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala LeuThr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser GlyLeu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser LeuGly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser AsnThr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 ThrHis Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val HisAsn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr TyrArg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu AsnGly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu ProAla Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro ArgGlu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu ThrLys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr ProSer Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu AsnAsn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly SerPhe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp GlnGln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu HisAsn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys 17214 PRT Artificial mouse anti-human monoclonal antibody 17 Asp Ile ValMet Thr Gln Ser Gln Arg Phe Met Ser Thr Thr Val Gly 1 5 10 15 Asp ArgVal Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Val Ser Ala 20 25 30 Val AlaTrp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr SerAla Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser GlySer Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Met Gln Ser 65 70 75 80 GluAsp Leu Ala Asp Phe Phe Cys Gln Gln Tyr Ser Asn Tyr Pro Trp 85 90 95 ThrPhe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr SerLeu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys HisLys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser ProVal Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 18 449 PRTArtificial mouse anti-human monoclonal antibody 18 Asp Val Lys Leu ValGlu Ser Gly Gly Gly Leu Val Lys Leu Gly Gly 1 5 10 15 Ser Leu Lys LeuSer Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Tyr Met Ser TrpVal Arg Gln Thr Pro Glu Lys Arg Leu Glu Leu Val 35 40 45 Ala Ala Ile AsnSer Asp Gly Gly Ile Thr Tyr Tyr Leu Asp Thr Val 50 55 60 Lys Gly Arg PheThr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln MetSer Ser Leu Lys Ser Glu Asp Thr Ala Leu Phe Tyr Cys 85 90 95 Ala Arg HisArg Ser Gly Tyr Phe Ser Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr SerVal Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 ProLeu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val ProSer 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn HisLys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys SerCys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu LeuLeu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro LysAsp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val ValVal Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp TyrVal Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg GluGlu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr ValLeu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys LysVal Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile SerLys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu ProPro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 CysLeu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met HisGlu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu SerPro Gly 435 440 445 Lys 19 5 PRT Artificial mouse anti-human monoclonalantibody 19 Glu Glu Gln Tyr Asn 1 5 20 5 PRT Artificial mouse anti-humanmonoclonal antibody 20 Val Ser Ile Thr Cys 1 5 21 5 PRT Artificial mouseanti-human monoclonal antibody 21 Leu Ile Val Ser Leu 1 5 22 10 PRTArtificial mouse anti-human monoclonal antibody 22 Ser Gly Thr Ala SerVal Val Cys Leu Leu 1 5 10 23 5 PRT Artificial mouse anti-humanmonoclonal antibody 23 Thr Lys Pro Arg Glu 1 5

1. Recombinant vector system comprising at least one copy of a nucleicacid encoding the antigen-binding site of the heavy chain of an antibodycomprising a nucleotide sequence encoding the CDR3 region (designatedH3), or/and encoding the CDR2 region (designated H2), or/and encodingthe CDR1 region (designated H1), as shown in FIG. 1 or/and FIG. 6, andat least one copy of a nucleic acid encoding the antigen-binding site ofthe light chain of an antibody comprising a nucleotide sequence encodingthe CDR3 region (designated L3), or/and encoding the CDR2 region(designated L2), or/and encoding the CDR1 region (designated L1), asshown in FIG. 1 or/and FIG. 6, wherein the nucleic acid encoding theantigen-binding site of the heavy chain and of the light chain haveseparate expression control sequences.
 2. Recombinant vector systemaccording to claim 1 comprising a first recombinant vector comprising atleast one copy of a nucleic acid encoding the antigen-binding site ofthe heavy chain and a second recombinant vector comprising at least onecopy of a nucleic acid encoding the antigen-binding site of the lightchain.
 3. Recombinant vector system according to claim 1 wherein atleast one copy of the nucleic acid encoding the antigen-binding site ofthe heavy chain and of the light chain are located on the samerecombinant vector.
 4. Method for the recombinant production of apolypeptide having an antigen-binding site comprising: (a) providing arecombinant vector system according to claim 1, (b) introducing therecombinant vector system into a suitable host cell, (c) culturing thehost cell under suitable conditions in a medium whereby an expression ofthe polypeptide takes place and (d) obtaining the expressed product fromthe medium and/or the host cell.
 5. The method of claim 4, wherein thehost cell is a mammalian cell.
 6. The method of claim 4, wherein betweensteps (a) and (b) a modification of the vector system takes placewherein the modification substantially does not alter the amino acidsequence of the antigen-binding site of the polypeptide to be expressed.7. The method of claim 4 further comprising preparing a diagnostic ortherapeutic agent from the expressed product.
 8. The method of claim 7,wherein the expressed product is coupled to a diagnostic marker.
 9. Themethod of claim 7, wherein the expressed product is coupled to acytotoxic agent.
 10. The method of claim 4, wherein the expressedproduct is selected from antibodies and antibody fragments.